Methods and compositions for expression of transgenes in plants

ABSTRACT

Transgenic plants are provided comprising a plurality of transgenes comprised in a single locus. In certain aspects, 7 or more transgenes may be expressed from a first locus. Methods are provided for transformation of plant cells with a plurality of transgenes. Also provided are methods for expressing and enhancing the expression of one or more transgenes in a plant.

This application is a continuation of co-pending U.S. application Ser.No. 12/910,377, filed Oct. 22, 2010; which claims the priority of U.S.Provisional Application No. 61/254,586, filed Oct. 23, 2009, the entiredisclosures of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates to the field of molecular biology. Morespecifically, the invention relates to transgenic plants comprisingmultiple transgenes and methods for expressing a plurality of transgenesin plants.

Description of the Related Art

One of the goals of genetic engineering is to produce hosts, such asplants, with important characteristics or traits. Recent advances ingenetic engineering have provided the requisite tools to transform hoststo contain and express foreign genes. Particularly desirable traits ofinterest for genetic engineering would include but are not limited toprotein production, resistance to insects and other pests anddisease-causing agents, tolerances to herbicides, enhanced stability,yield, or shelf-life, environmental tolerances, and nutritionalenhancements.

The technological advances in transformation and regeneration haveenabled researchers to take segments of DNA, such as a gene or genesfrom a heterologous source, or native source and incorporate theexogenous DNA into a host's genome. The gene or gene(s) can then beexpressed in the host cell to exhibit the added characteristic(s) ortrait(s). In most transformation approaches, a single vector containing1-2 genes conferring desirable characteristic(s) is introduced into ahost of interest via an appropriate expression vector. Expression of agreater number of transgenes in host cells and organisms has proven tobe costly and time consuming.

SUMMARY OF THE INVENTION

In a first aspect, there is provided a transgenic plant comprising afirst locus comprising a plurality of transgenes operably linked topromoter sequences. For example, a locus may comprise a plurality ofexpression cassettes for the expression of a plurality of transgenes. Incertain embodiments, a first locus comprises at least 6, at least 7, atleast 8, at least 9, at least 10, or more, transgenes.

In certain aspects, a transgenic plant according to the inventioncomprises enhanced expression of at least one transgene relative to anotherwise isogenic transgenic plant wherein the same transgene isintroduced in a locus comprising fewer total transgenes than a locus ina plant according to the invention. In further embodiments, a plantaccording to the invention comprises enhanced expression of at least 2,3, 4, 5 or more transgenes relative to a second, otherwise isogenic,transgenic plant wherein the corresponding transgenes are comprised in alocus in the second plant that comprises fewer total transgenes than aplant according to the invention.

In further aspects, a plant according to the invention comprises a firstlocus comprising a plurality of transgenes wherein at least 1, 2, 3, 4,5, 6, 7, 8, 9 or more of the transgenes confers a trait of agronomicinterest when expressed in a plant. For example, a transgene may conferherbicide tolerance (e.g., glyphosate or other herbicide tolerance),drought resistance, insect resistance, fungus resistance, virusresistance, bacteria resistance, male sterility, cold tolerance, salttolerance, increased yield, enhanced oil composition, increased oilcontent, enhanced nutrient use efficiency or altered amino acid contentto a plant expressing the transgene. In certain embodiments, a firstlocus comprises a plurality of transgenes that confer traits ofagronomic interest. For example, a first locus may comprise at least onetransgene that confers herbicide tolerance, at least one transgene thatconfers insect resistance and/or at least one transgene that confersdrought tolerance. In a further embodiment, a first locus comprises atleast two transgenes that confer herbicide tolerance, at least twotransgenes that confer insect resistance and/or at least two transgenesthat confer drought tolerance. In still a further embodiment, a firstlocus comprises at least one transgene that confers above-ground insectresistance and at least one transgene that confers below-ground insectresistance.

A variety of transgenes are known in art and may be used in accordancewith the invention. For example, one or more transgenes may be selectedfrom those disclosed in International (PCT) Publn. WO 2008/063755, thecontents of which are incorporated herein by reference in theirentirety. Examples, of transgenes for use according to the inventioninclude, but are not limited to, transgenes conferring herbicidetolerance, drought tolerance or insect tolerance.

In further aspects, a transgenic plant according to the invention is amonocotyledonous plant such as, wheat, maize, rye, rice, corn, oat,barley, turfgrass, sorghum, millet or sugarcane. In certain otherembodiments, a transgenic plant is a dicotyledonous plant, such astobacco, tomato, potato, soybean, cotton, canola, sunflower or alfalfa.

In certain aspects, the invention provides a part of a transgenic plantdescribed herein, such as a protoplast, cell, gamete, meristem, root,pistil, anther, flower, seed, embryo, stalk or petiole. In certainembodiments, seed of a transgenic plant described herein is providedwherein the seed comprises the first locus comprising a plurality oftransgenes. In a further embodiment, there is provided a progeny plantof a transgenic plant according to the invention wherein the progenyplant comprises the first locus comprising a plurality of transgenes. Instill a further embodiment, there is provided a method for producing acommercial product comprising obtaining a plant according to theinvention or part thereof and producing a commercial product therefrom.

In a further aspect, the invention provides a tissue culture ofregenerable cells of a transgenic plant according to the invention. Forexample, the regenerable cells may be from embryos, meristematic cells,pollen, leaves, roots, root tips, anther, pistil, flower, seed, boll orstem. In still a further embodiment, there is provided a plantregenerated from the a tissue culture according to the invention.

In still a further aspect, there is provided a method of transforming aplant cell comprising introducing a plurality of transgenes into a plantcell, wherein the plurality of transgene are comprised on a single DNAmolecule. For example, a transformation method may comprise introducingat least 6, 7, 8, 9 or 10 transgenes into a plant cell wherein thetransgenes are comprised on a single DNA molecule. In a furtherembodiment, a transformation method comprises selecting a transformedplant cell wherein said cell comprises the plurality of transgenes in asingle-copy transformation event (e.g., a transformation event that isfree from backbone vector sequence).

In yet a further aspect, there is provided a method for expressing aplurality of transgenes in a plant comprising expressing said pluralityof transgenes in the plant wherein the transgenes are comprised in asingle locus said event comprising a plurality transgenes operablylinked to promoter sequences.

In a further aspect, there is provided a method for enhancing theexpression of at least a first transgene in a plant comprisingexpressing the transgene in the plant according to the invention. Forexample, transgene expression may be enhanced by expressing a transgenecomprised in a locus wherein the locus comprises a plurality ofadditional transgenes. In certain embodiments, a plant comprises atleast 5, 6, 7, 8, 9 or 10 additional transgenes. In a furtherembodiment, a method for enhancing expression of at least one transgenecomprises enhancing expression of the transgene relative to an otherwiseisogenic plant wherein the transgene is comprised in a locus thatcomprises fewer transgenes than the locus in a plant according to theinvention. For example, an expressed transgene may be comprised in alocus comprising at least 5, 6, 7, 8 or 9 additional transgenes and itsexpression enhanced relative to expression of the same transgene from anotherwise isogenic plant comprising a locus with fewer additionaltransgenes (e.g., less than 5, 6, 7, 8 or 9 additional transgenes).

In still further aspects, a method for enhancing expression of at leastone transgene in a plant transformed to contain a plurality oftransgenes comprises enhancing expression of a transgene that confersherbicide tolerance, drought tolerance or insect tolerance. In stillfurther embodiments, a method according to the invention may be definedas a method of enhancing the expression of two or more transgeneswherein the transgenes are comprised in a first transgenic eventcomprising a plurality of additional transgenes.

In certain aspects, two or more transgenes (e.g., a plurality oftransgenes) are arranged in tandem. In one embodiment, tandem refers toan arrangement in which there is a substantial absence of interveningDNA between transgenes. In specific embodiments, tandem refers to lackof a length of intervening sequence that, if present, interferes withtransgene expression and/or the ability to transform a plurality oftransgenes into plants.

As used herein the terms “encode” or “encoding” with reference to anucleic acid are used to make the invention readily understandable bythe skilled artisan however these terms may be used interchangeably with“comprise” or “comprising” respectively.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the invention. Theinvention may be better understood by reference to one or more of thesedrawings in combination with the detailed description of specificembodiments presented herein:

FIG. 1A-B: Vector schematics. FIG. 1A, illustrates the binary T-DNAvector used for recombination in vector construction. FIG. 1B, providesa schematic of the 10 gene expression cassette.

FIG. 2: A Southern blot analysis of the plant lines transformed with the10 gene vector.

FIG. 3: FISH analysis of the chromosomes from three lines transformedwith the 10 gene vector.

FIG. 4: Graphs illustrate RNA expression from various genes in 10 genevector transformed lines. Horizontal lines indict the level of controlexpression from genes not comprised in the 10 gene system.

FIG. 5: Graphs indicate pest mortality when exposed to plantstransformed with the 10 gene vector compared to control plants.

FIG. 6: Graphs indicate leaf damage in plants transformed with the 10gene vector exposed to pests as compared to control plants.

FIG. 7: Graphs indicate root damage in plants transformed with the 10gene vector exposed to pests as compared to control plants.

DETAILED DESCRIPTION OF THE INVENTION

In certain aspects, the invention provides methods and compositionsrelated to expression of multiple transgenes in plants from a singlelocus. For example, there is provided a method of transforming a plantcell with a vector comprising a plurality of transgenes and regeneratinga plant therefrom. In another aspect, transgenic plants are providedthat comprise a first locus comprising a plurality of transgenes linkedto expression control sequences. In certain aspects, there is provided amethod for expression of a plurality of transgenes in a transgenic plantcomprising expressing a plurality of transgenes from a first locuscomprising a plurality of transgenes linked to expression controlsequences. In still a further aspect, there is provided a method forenhancing expression of one or more transgenes comprising expressingsaid one or more transgenes from a first locus comprising a plurality oftransgenes.

In certain aspects, methods and plants according to the inventionconcern expression of plurality of transgenes comprised in a singlelocus that confer a plurality of traits of agronomic interest. In oneembodiment, a locus comprises one or more transgenes that confer aherbicide tolerance trait and three or more transgenes that conferinsect resistance traits. In another embodiment, a locus comprises oneor more transgenes that confer a herbicide tolerance trait, two or moreof the insect resistance traits, and one or more transgenes that confera drought tolerance trait.

In still another aspect a locus comprises a transgene that confers malesterility. For example, a locus may comprise one or more transgenes thatconfer a herbicide tolerance trait, two or more transgenes that conferinsect resistance traits, one or more transgenes that confer a droughttolerance trait, and one or more transgenes that confer a male sterilitytrait. In another embodiment, a locus comprises one or more transgenesthat confer a herbicide tolerance trait, two or more transgenes thatconfer insect resistance traits, one or more transgenes that confer anenhanced amino acid content trait, one or more transgenes that confer adrought tolerance trait, and one or more transgenes that confer a malesterility trait.

In a further aspect, a locus may comprise a transgene that confers anincreased yield trait. For instance, a locus may comprise one or moretransgenes that confer a herbicide tolerance trait, two or moretransgenes that confer insect resistance traits, one or more transgenesthat confer a drought tolerance trait, one or more transgenes thatconfer a male sterility trait, and one or more transgenes that confer anincreased yield trait. In another embodiment, a locus comprises one ormore transgenes that confer herbicide tolerance trait, one or moretransgenes that confer an insect resistance trait, one or moretransgenes that confer an enhanced amino acid content trait, one or moretransgenes that confer a drought tolerance trait, one or more transgenesthat confer a male sterility trait, and one or more transgenes thatconfer an enhanced yield trait.

In still a further aspect, a locus may comprise a transgene that confersa nutrient use efficiency trait. For example, a locus may comprise oneor more transgenes that confer a herbicide tolerance trait, two or moretransgenes that confer insect resistance traits, one or more transgenesthat confer drought tolerance traits, one or more transgenes that confera the male sterility trait, one or more transgenes that confer anenhanced yield trait, and one or more transgenes that confer a nutrientuse efficiency trait.

In yet further aspects, a locus may comprise a transgene that confers acold tolerance trait. In one example, a locus may comprise one or moretransgenes that confer a herbicide tolerance trait, two or moretransgenes that confer insect resistance traits, one or more transgenesthat confer a drought tolerance trait, one or more transgenes thatconfer a male sterility trait, one or more transgenes that confer anenhanced yield trait, one or more transgenes that confer a nutrient useefficiency trait and one or more transgenes that confer a cold tolerancetrait. In another embodiment, a locus comprises one or more transgenesthat confer a herbicide tolerance trait, one or more transgenes thatconfer an insect resistance trait, one or more transgenes that confer anenhanced amino acid content trait, one or more transgenes that confer adrought tolerance trait, one or more transgenes that confer a malesterility trait, one or more transgenes that confer an enhanced yieldtrait, one or more transgenes that confer a nutrient use efficiencytrait, one or more transgenes that confer an enhanced oil content trait,one or more transgenes that confer an enhanced protein content trait,and one or more transgenes that confer a cold tolerance trait.

In certain aspects, a locus may comprise a transgene that confers anenhanced amino acid content trait. In another embodiment, a locuscomprises one or more transgenes that confer a herbicide tolerancetrait, three or more transgenes that confer insect resistance traits,and one or more transgenes that confer an enhanced amino acid content.In another embodiment, a locus comprises one or more transgenes thatconfer a herbicide tolerance trait, two or more transgenes that conferinsect resistance traits, one or more transgenes that confer an enhancedamino acid content trait, and one or more transgenes that confer adrought tolerance trait.

In further aspects, a locus may comprise a transgene that confers anenhanced oil content trait. For example, a locus may comprise one ormore transgenes that confer a herbicide tolerance trait, two or moretransgenes that confer insect resistance traits, one or more transgenesthat confer an enhanced amino acid content trait, one or more transgenesthat confer a drought tolerance trait, one or more transgenes thatconfer a male sterility trait, one or more transgenes that confer anenhanced yield trait, and one or more transgenes that confer an enhancedoil content trait. In another embodiment, a locus comprises one or moretransgenes that confer a herbicide tolerance trait, two or moretransgenes that confer insect resistance traits, one or more transgenesthat confer an enhanced amino acid content trait, one or more transgenesthat confer a drought tolerance trait, one or more transgenes thatconfer a male sterility trait, one or more transgenes that confer anenhanced yield trait, one or more transgenes that confer a nutrient useefficiency trait, one or more transgenes that confer an enhanced oilcontent trait, one or more transgenes that confer an enhanced proteincontent trait, and one or more transgenes that confer a cold tolerancetrait.

I. PLANT TRANSFORMATION AND TRANSGENE EXPRESSION CONSTRUCTS

Certain embodiments of the current invention concern planttransformation constructs. In certain embodiments of the invention,transgene coding sequences are provided operably linked to a promoter(e.g., a heterologous promoter), in either sense or antisenseorientation. Expression constructs are also provided comprising thesesequences, as are plants and plant cells transformed with the sequences.

The construction of vectors which may be employed in conjunction withplant transformation techniques using these or other sequences accordingto the invention will be known to those of skill of the art in light ofthe present disclosure (see, for example, Sambrook et al., 1989; Gelvinet al., 1990). The techniques of the current invention are thus notlimited to any particular nucleic acid sequences.

Particularly useful for transformation are expression cassettes whichhave been isolated from such vectors. DNA segments used for transformingplant cells will, of course, generally comprise the RNA coding sequence,cDNA, gene or genes which one desires to introduce into and haveexpressed in the host cells. These DNA segments can further includestructures such as promoters, enhancers, polylinkers, or even regulatorygenes as desired. The DNA segment or gene chosen for cellularintroduction will often encode a protein which will be expressed in theresultant recombinant cells resulting in a screenable or selectabletrait and/or which will impart an improved phenotype to the resultingtransgenic plant. However, this may not always be the case, and thepresent invention also encompasses transgenic plants incorporatingnon-expressed transgenes. Preferred components likely to be includedwith vectors used in the current invention are as follows.

A. Regulatory Elements

Exemplary promoters for expression of a transgene include plant promotersuch as the CaMV 35S promoter (Odell et al., 1985), or others such asCaMV 19S (Lawton et al., 1987), nos (Ebert et al., 1987), Adh (Walker etal., 1987), sucrose synthase (Yang and Russell, 1990), a-tubulin, actin(Wang et al., 1992), cab (Sullivan et al., 1989), PEPCase (Hudspeth andGrula, 1989) or those associated with the R gene complex (Chandler etal., 1989). Tissue specific promoters such as root cell promoters(Conkling et al., 1990) and tissue specific enhancers (Fromm et al.,1986) are also contemplated to be particularly useful, as are induciblepromoters such as ABA- and turgor-inducible promoters. In certainaspects, a promoter for use according to the invention is a ePCISV,TubA, eFMV, FMV, e35S, 35S or Ract1 promoter.

In certain aspects, transformation events comprised in transgenic plantsaccording to the invention comprise a plurality of promoter sequences.In certain aspects, a promoter sequence is repeated no more than about2, 3, 4, or 5 times in a single transformation event. In otherembodiments, identical or highly homologous promoter sequences arelinked to at least 2, 3, 4, 5 or more transgenes in a singletransformation event. In certain embodiments, a transformation eventcomprising a plurality of transgenes comprises at least 2, 3, 4, 5, 6,7, 8, 9 or 10 different promoter sequences.

In further embodiments, identical or highly homologous promotersequences are linked to transgenes that confer similar traits (e.g.,transgenes that confer insect resistance). In certain aspects, two ormore identical or highly homologous promoter sequences are separated byat least 1, 2 or 3 expression cassettes within a single transformationevent. In other embodiments, identical or highly homologous promotersequences are linked to two or more contiguous expression cassettes in asingle transformation event.

The DNA sequence between the transcription initiation site and the startof the coding sequence, i.e., the untranslated leader sequence, can alsoinfluence gene expression. One may thus wish to employ a particularleader sequence with a transformation construct of the invention.Preferred leader sequences are contemplated to include those whichcomprise sequences predicted to direct optimum expression of theattached gene, i.e., to include a preferred consensus leader sequencewhich may increase or maintain mRNA stability and prevent inappropriateinitiation of translation. The choice of such sequences will be known tothose of skill in the art in light of the present disclosure. Sequencesthat are derived from genes that are highly expressed in plants willtypically be preferred.

It is specifically envisioned that transgene coding sequences may beintroduced under the control of novel promoters or enhancers, etc., orhomologous or tissue specific promoters or control elements. Vectors foruse in tissue-specific targeting of genes in transgenic plants willtypically include tissue-specific promoters and may also include othertissue-specific control elements such as enhancer sequences. Promoterswhich direct specific or enhanced expression in certain plant tissueswill be known to those of skill in the art in light of the presentdisclosure. These include, for example, the rbcS promoter, specific forgreen tissue; the ocs, nos and mas promoters which have higher activityin roots or wounded leaf tissue; a truncated (−90 to +8) 35S promoterwhich directs enhanced expression in roots, and an α-tubulin gene thatalso directs expression in roots.

B. Terminators

Transformation constructs prepared in accordance with the invention willtypically include a 3′ end DNA sequence that acts as a signal toterminate transcription and allow for the poly-adenylation of the mRNAproduced by coding sequences operably linked to a transgene. Terminatorswhich are deemed to be particularly useful in this context include thosefrom the nopaline synthase gene of Agrobacterium tumefaciens (nos 3′end) (Bevan et al., 1983), the terminator for the T7 transcript from theoctopine synthase gene of Agrobacterium tumefaciens, and the 3′ end ofthe protease inhibitor I or II genes from potato or tomato. Regulatoryelements such as an Adh intron (Callis et al., 1987), sucrose synthaseintron (Vasil et al., 1989) or TMV omega element (Gallie et al., 1989),may further be included where desired. In certain aspects, a terminatorfor use according to the invention is a Hsp17, TubA, Ara5, 35S, nos orTr7 terminator.

In certain aspects, transformation events comprised in transgenic plantsaccording to the invention comprise a plurality of terminator sequences.In certain aspects, a terminator sequence is repeated no more than about2, 3, 4, or 5 times in a single transformation event. In otherembodiments, identical or highly homologous terminator sequences arelinked to at least 2, 3, 4, 5 or more transgenes in a singletransformation event. In certain embodiments, a transformation eventcomprising a plurality of transgenes comprises at least 2, 3, 4, 5, 6,7, 8, 9 or 10 different terminator sequences. In further embodiments,identical or highly homologous terminator sequences are linked totransgenes that confer similar traits (e.g., transgenes that conferinsect resistance). In certain aspects, two or more identical or highlyhomologous terminator sequences are separated by at least 1, 2 or 3expression cassettes with a single transformation event. In otherembodiments, identical or highly homologous terminator sequences arelinked to two or more contiguous expression cassettes in a singletransformation event.

C. Intron Sequences

In certain aspects, intron sequences are included an expression cassetteand may enhance transgene expression. In certain aspects, an intron foruse according to the invention is a Ract1, TubA, Sus1 or Hsp70 intron.

In certain aspects, transformation events comprised in transgenic plantsaccording to the invention comprise a plurality of intron sequences. Incertain aspects, an intron sequence is repeated no more than about 2, 3,4, or 5 times in a single transformation event. In other embodiments,identical or highly homologous intron sequences are linked to at least2, 3, 4, 5 or more transgenes in a single transformation event. Incertain embodiments, a transformation event comprising a plurality oftransgenes comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 differentintron sequences.

In further embodiments, identical or highly homologous intron sequencesare linked to transgenes that confer similar traits (e.g., transgenesthat confer insect resistance). In certain aspects, two or moreidentical or highly homologous intron sequences are separated by atleast 1, 2 or 3 expression cassettes within a single transformationevent. In other embodiments, identical or highly homologous intronsequences are linked to two or more contiguous expression cassettes in asingle transformation event.

D. Transit or Signal Peptides

Sequences that are joined to the coding sequence of an expressed gene,which are removed post-translationally from the initial translationproduct and which facilitate the transport of the protein into orthrough intracellular or extracellular membranes, are termed transit(usually into vacuoles, vesicles, plastids and other intracellularorganelles) and signal sequences (usually to the endoplasmic reticulum,golgi apparatus and outside of the cellular membrane). By facilitatingthe transport of the protein into compartments inside and outside thecell, these sequences may increase the accumulation of gene productprotecting them from proteolytic degradation. These sequences also allowfor additional mRNA sequences from highly expressed genes to be attachedto the coding sequence of the genes. Since mRNA being translated byribosomes is more stable than naked mRNA, the presence of translatablemRNA in front of the gene may increase the overall stability of the mRNAtranscript from the gene and thereby increase synthesis of the geneproduct. Since transit and signal sequences are usuallypost-translationally removed from the initial translation product, theuse of these sequences allows for the addition of extra translatedsequences that may not appear on the final polypeptide. It further iscontemplated that targeting of certain proteins may be desirable inorder to enhance the stability of the protein (U.S. Pat. No. 5,545,818,incorporated herein by reference in its entirety).

Additionally, vectors may be constructed and employed in theintracellular targeting of a specific gene product within the cells of atransgenic plant or in directing a protein to the extracellularenvironment. This generally will be achieved by joining a DNA sequenceencoding a transit or signal peptide sequence to the coding sequence ofa particular gene. The resultant transit, or signal, peptide willtransport the protein to a particular intracellular, or extracellulardestination, respectively, and will then be post-translationallyremoved.

E. Marker Genes

By employing a selectable or screenable marker protein, one can provideor enhance the ability to identify transformants. “Marker genes” aregenes that impart a distinct phenotype to cells expressing the markerprotein and thus allow such transformed cells to be distinguished fromcells that do not have the marker. Such genes may encode either aselectable or screenable marker, depending on whether the marker confersa trait which one can “select” for by chemical means, i.e., through theuse of a selective agent (e.g., a herbicide, antibiotic, or the like),or whether it is simply a trait that one can identify throughobservation or testing, i.e., by “screening” (e.g., the greenfluorescent protein). Of course, many examples of suitable markerproteins are known to the art and can be employed in the practice of theinvention.

Included within the terms “selectable” or “screenable markers” also aregenes which encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers which are secretable antigens that can be identified byantibody interaction, or even secretable enzymes which can be detectedby their catalytic activity. Secretable proteins fall into a number ofclasses, including small, diffusible proteins detectable, e.g., byELISA; small active enzymes detectable in extracellular solution (e.g.,α-amylase, β-lactamase, phosphinothricin acetyltransferase); andproteins that are inserted or trapped in the cell wall (e.g., proteinsthat include a leader sequence such as that found in the expression unitof extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene thatencodes a protein that becomes sequestered in the cell wall, and whichprotein includes a unique epitope is considered to be particularlyadvantageous. Such a secreted antigen marker would ideally employ anepitope sequence that would provide low background in plant tissue, apromoter-leader sequence that would impart efficient expression andtargeting across the plasma membrane, and would produce protein that isbound in the cell wall and yet accessible to antibodies. A normallysecreted wall protein modified to include a unique epitope would satisfyall such requirements.

Many selectable marker coding regions are known and could be used withthe present invention including, but not limited to, neo (Potrykus etal., 1985), which provides kanamycin resistance and can be selected forusing kanamycin, G418, paromomycin, etc.; bar, which confers bialaphosor phosphinothricin resistance; a mutant EPSP synthase protein (Hincheeet al., 1988) conferring glyphosate resistance; a nitrilase such as bxnfrom Klebsiella ozaenae which confers resistance to bromoxynil (Stalkeret al., 1988); a mutant acetolactate synthase (ALS) which confersresistance to imidazolinone, sulfonylurea or other ALS inhibitingchemicals (European Patent Application 154,204, 1985); a methotrexateresistant DHFR (Thillet et al., 1988), a dalapon dehalogenase thatconfers resistance to the herbicide dalapon; or a mutated anthranilatesynthase that confers resistance to 5-methyl tryptophan.

An illustrative embodiment of selectable marker capable of being used insystems to select transformants are those that encode the enzymephosphinothricin acetyltransferase, such as the bar gene fromStreptomyces hygroscopicus or the pat gene from Streptomycesviridochromogenes. The enzyme phosphinothricin acetyl transferase (PAT)inactivates the active ingredient in the herbicide bialaphos,phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami etal., 1986; Twell et al., 1989) causing rapid accumulation of ammonia andcell death.

Screenable markers that may be employed include a β-glucuronidase (GUS)or uidA gene which encodes an enzyme for which various chromogenicsubstrates are known; an R-locus gene, which encodes a product thatregulates the production of anthocyanin pigments (red color) in planttissues (Dellaporta et al., 1988); a β-lactamase gene (Sutcliffe, 1978),which encodes an enzyme for which various chromogenic substrates areknown (e.g., PADAC, a chromogenic cephalosporin); a xylE gene (Zukowskyet al., 1983) which encodes a catechol dioxygenase that can convertchromogenic catechols; an α-amylase gene (Ikuta et al., 1990); atyrosinase gene (Katz et al., 1983) which encodes an enzyme capable ofoxidizing tyrosine to DOPA and dopaquinone which in turn condenses toform the easily-detectable compound melanin; a β-galactosidase gene,which encodes an enzyme for which there are chromogenic substrates; aluciferase (lux) gene (Ow et al., 1986), which allows forbioluminescence detection; an aequorin gene (Prasher et al., 1985) whichmay be employed in calcium-sensitive bioluminescence detection; or agene encoding for green fluorescent protein (Sheen et al., 1995;Haseloff et al., 1997; Reichel et al., 1996; Tian et al., 1997; WO97/41228).

Another screenable marker contemplated for use in the present inventionis firefly luciferase, encoded by the lux gene. The presence of the luxgene in transformed cells may be detected using, for example, X-rayfilm, scintillation counting, fluorescent spectrophotometry, low-lightvideo cameras, photon counting cameras or multiwell luminometry. It alsois envisioned that this system may be developed for population screeningfor bioluminescence, such as on tissue culture plates, or even for wholeplant screening. The gene which encodes green fluorescent protein (GFP)is also contemplated as a particularly useful reporter gene (Sheen etal., 1995; Haseloff et al., 1997; Reichel et al., 1996; Tian et al.,1997; WO 97/41228). Expression of green fluorescent protein may bevisualized in a cell or plant as fluorescence following illumination byparticular wavelengths of light.

F. Example Transgenes

1. Male Sterility

Examples of genes conferring male sterility include those disclosed inU.S. Pat. No. 3,861,709, U.S. Pat. No. 3,710,511, U.S. Pat. No.4,654,465, U.S. Pat. No. 5,625,132, and U.S. Pat. No. 4,727,219, each ofthe disclosures of which are specifically incorporated herein byreference in their entirety. The use of herbicide-inducible malesterility genes is described in U.S. Pat. No. 6,762,344. Male sterilitygenes can increase the efficiency with which hybrids are made, in thatthey eliminate the need to physically emasculate plants used as a femalein a given cross.

2. Herbicide Tolerance

Numerous herbicide resistance genes are known and may be employed withthe invention. An example is a gene conferring resistance to a herbicidethat inhibits the growing point or meristem, such as an imidazalinone ora sulfonylurea. Exemplary genes in this category code for mutant ALS andAHAS enzyme as described, for example, by Lee et al., (1988); Gleen etal., (1992) and Miki et al., (1990).

Resistance genes for glyphosate (resistance conferred by mutant5-enolpyruvl-3 phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin-acetyl transferase (bar) genes) may alsobe used. See, for example, U.S. Pat. No. 4,940,835 to Shah, et al.,which discloses the nucleotide sequence of a form of EPSPS which canconfer glyphosate resistance. Examples of specific EPSPS transformationevents conferring glyphosate resistance are provided by U.S. Pat. No.6,040,497.

A DNA molecule encoding a mutant aroA gene can be obtained under ATCCaccession number 39256, and the nucleotide sequence of the mutant geneis disclosed in U.S. Pat. No. 4,769,061 to Comai. European patentapplication No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes which confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a phosphinothricin-acetyltransferase gene isprovided in European application No. 0 242 246 to Leemans et al. DeGreefet al., (1989), describe the production of transgenic plants thatexpress chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy propionic acids and cycloshexones, such as sethoxydim andhaloxyfop are the Acct-S1, Accl-S2 and Acct-S3 genes described byMarshall et al., (1992). In certain aspects, a DMO transgene may be usedto mediate dicamba tolerance.

Genes are also known conferring resistance to a herbicide that inhibitsphotosynthesis, such as a triazine (psbA and gs+ genes) and abenzonitrile (nitrilase gene). Przibila et al., (1991), describe thetransformation of Chlamydomonas with plasmids encoding mutant psbAgenes. Nucleotide sequences for nitrilase genes are disclosed in U.S.Pat. No. 4,810,648 to Stalker, and DNA molecules containing these genesare available under ATCC Accession Nos. 53435, 67441, and 67442. Cloningand expression of DNA coding for a glutathione S-transferase isdescribed by Hayes et al., (1992).

Other examples of herbicide resistance have been described, forinstance, in U.S. Pat. Nos. 6,803,501; 6,448,476; 6,248,876; 6,225,114;6,107,549; 5,866,775; 5,804,425; 5,633,435; 5,463,175.

3. Disease and Pest Resistance

Plant defenses are often activated by specific interaction between theproduct of a disease resistance gene (R) in the plant and the product ofa corresponding avirulence (Avr) gene in the pathogen. A plant line canbe transformed with cloned resistance gene to engineer plants that areresistant to specific pathogen strains. See, for example Jones et al.,(1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporiumfulvum); Martin et al., (1993) (tomato Pto gene for resistance toPseudomonas syringae pv.); and Mindrinos et al., (1994) (ArabidopsisRSP2 gene for resistance to Pseudomonas syringae).

A viral-invasive protein or a complex toxin derived therefrom may alsobe used for viral disease resistance. For example, the accumulation ofviral coat proteins in transformed plant cells imparts resistance toviral infection and/or disease development effected by the virus fromwhich the coat protein gene is derived, as well as by related viruses.See Beachy et al., (1990). Coat protein-mediated resistance has beenconferred upon transformed plants against alfalfa mosaic virus, cucumbermosaic virus, tobacco streak virus, potato virus X, potato virus Y,tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.

A virus-specific antibody may also be used. See, for example,Tavladoraki et al., (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack. Virusresistance has also been described in, for example, U.S. Pat. Nos.6,617,496; 6,608,241; 6,015,940; 6,013,864; 5,850,023 and 5,304,730.

Logemann et al., (1992), for example, disclose transgenic plantsexpressing a barley ribosome-inactivating gene have an increasedresistance to fungal disease. Other examples of fungal diseaseresistance are provided in U.S. Pat. Nos. 6,653,280; 6,573,361;6,506,962; 6,316,407; 6,215,048; 5,516,671; 5,773,696; 6,121,436;6,316,407; and 6,506,962).

Nematode resistance has been described, for example, in U.S. Pat. No.6,228,992 and bacterial disease resistance in U.S. Pat. No. 5,516,671.

4. Insect Resistance

One example of an insect resistance gene includes a Bacillusthuringiensis protein, a derivative thereof or a synthetic polypeptidemodeled thereon. See, for example, Geiser et al., (1986), who disclosethe cloning and nucleotide sequence of a Bt δ-endotoxin gene. Moreover,DNA molecules encoding δ-endotoxin genes can be purchased from theAmerican Type Culture Collection, Manassas, Va., for example, under ATCCAccession Nos. 40098, 67136, 31995 and 31998. Another example is alectin. See, for example, Van Damme et al., (1994), who disclose thenucleotide sequences of several Clivia miniata mannose-binding lectingenes. A vitamin-binding protein may also be used, such as avidin. SeePCT application US93/06487, the contents of which are herebyincorporated by reference. This application teaches the use of avidinand avidin homologues as larvicides against insect pests.

Yet another insect resistance gene is an enzyme inhibitor, for example,a protease or proteinase inhibitor or an amylase inhibitor. See, forexample, Abe et al., (1987) (nucleotide sequence of rice cysteineproteinase inhibitor), Huub et al., (1993) (nucleotide sequence of cDNAencoding tobacco proteinase inhibitor I), and Sumitani et al., (1993)(nucleotide sequence of Streptomyces nitrosporeus α-amylase inhibitor).An insect-specific hormone or pheromone may also be used. See, forexample, the disclosure by Hammock et al., (1990), of baculovirusexpression of cloned juvenile hormone esterase, an inactivator ofjuvenile hormone.

Still further nucleic acids encoding proteins that confer insectresistance can be derived from a number of organisms that include, butare not limited to, Bacillus thuringiensis, Xenorhabdus sp., orPhotorhabdus sp. For example, construct or transgenic plants maycomprise one or more B. thuringiensis proteins toxic to an insectspecies or multiple insect species (e.g., Cry1Aa, Cry1Ab, Cry1Ac,Cry1Ba, CryBb, Cry1Ca, Cry1F, Cry2Aa, Cry2Ab, Cry3A, Cry3B, Cry3C, Cry9,Cry34 or Cry 35). Use of multiple genes to confer insect resistance maydelay the onset of resistance in a population of an otherwisesusceptible insect species to one or more of the insecticidal nucleicacids expressed within the transgenic plant. Alternatively, expressionof a B. thuringiensis insecticidal protein toxic to a particular targetinsect pest along with a different proteinaceous agent toxic to the sameinsect pest but which confers toxicity by a means different from thatexhibited by the B. thuringiensis toxin is desirable. Such otherdifferent proteinaceous agents may comprise any of Cry insecticidalproteins, Cyt insecticidal proteins, insecticidal proteins fromXenorhabdus sp. or Photorhabdus sp., B. thuringiensis vegetativeinsecticidal proteins, and the like. Examples of such proteins encodedby insect toxin genes includes, but are not limited to, ET29, TIC809,TIC810, TIC105, TIC127, TIC128, TIC812 and ET37 (WO 07/027776), TIC807,AXMI-027, AXMI-036, and AXMI-038 (WO 06/107761), AXMI-018, AXMI-020, andAXMI-021 (WO 06/083891), AXMI-O1O (WO 05/038032), AXMI-003 (WO05/021585), AXMI-008 (US 2004/0250311), AXMI-006 (US 2004/0216186),AXMI-007 (US 2004/0210965), AXMI-009 (US 2004/0210964), AXMI-014 (US2004/0197917), AXMI-004 (US 2004/0197916), AXMI-028 and AXMI-029 (WO06/119457) and AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 andAXMI-004 (WO 04/074462). All of the foregoing references areincorporated herein in their entirety.

Additional nucleic acids that can be used to confer insect resistancemay encode RNA for gene suppression of an essential gene in the insectpest. For example, a nucleic acid may comprise an antisense or dsRNAsequence for suppression of an insect Dv49 gene.

Still other examples include an insect-specific antibody or animmunotoxin derived therefrom and a developmental-arrestive protein. SeeTaylor et al., (1994), who described enzymatic inactivation intransgenic tobacco via production of single-chain antibody fragments.Numerous other examples of insect resistance have been described. See,for example, U.S. Pat. Nos. 6,809,078; 6,713,063; 6,686,452; 6,657,046;6,645,497; 6,642,030; 6,639,054; 6,620,988; 6,593,293; 6,555,655;6,538,109; 6,537,756; 6,521,442; 6,501,009; 6,468,523; 6,326,351;6,313,378; 6,284,949; 6,281,016; 6,248,536; 6,242,241; 6,221,649;6,177,615; 6,156,573; 6,153,814; 6,110,464; 6,093,695; 6,063,756;6,063,597; 6,023,013; 5,959,091; 5,942,664; 5,942,658, 5,880,275;5,763,245 and 5,763,241.

5. Modified Fatty Acid, Phytate and Carbohydrate Metabolism

Genes may be used conferring modified fatty acid metabolism, in terms ofcontent and quality. For example, stearyl-ACP desaturase genes may beused. See Knutzon et al., (1992). Various fatty acid desaturases havealso been described, such as a Saccharomyces cerevisiae OLE1 geneencoding Δ9-fatty acid desaturase, an enzyme which forms themonounsaturated palmitoleic (16:1) and oleic (18:1) fatty acids frompalmitoyl (16:0) or stearoyl (18:0) CoA (McDonough et al., 1992); a geneencoding a stearoyl-acyl carrier protein delta-9 desaturase from castor(Fox et al. 1993); Δ6- and Δ12-desaturases from the cyanobacteriaSynechocystis responsible for the conversion of linoleic acid (18:2) togamma-linolenic acid (18:3 gamma) (Reddy et al. 1993); a gene fromArabidopsis thaliana that encodes an omega-3 desaturase (Arondel et al.1992)); plant Δ9-desaturases (PCT Application Publ. No. WO 91/13972) andsoybean and Brassica Δ15 desaturases (European Patent Application Publ.No. EP 0616644).

Modified oils production is disclosed, for example, in U.S. Pat. Nos.6,444,876; 6,426,447 and 6,380,462. High oil production is disclosed,for example, in U.S. Pat. Nos. 6,495,739; 5,608,149; 6,483,008 and6,476,295. Modified fatty acid content is disclosed, for example, inU.S. Pat. Nos. 6,828,475; 6,822,141; 6,770,465; 6,706,950; 6,660,849;6,596,538; 6,589,767; 6,537,750; 6,489,461 and 6,459,018.

Phytate metabolism may also be modified by introduction of aphytase-encoding gene to enhance breakdown of phytate, adding more freephosphate to the transformed plant. For example, see Van Hartingsveldtet al., (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. In corn, this, for example, could beaccomplished by cloning and then reintroducing DNA associated with thesingle allele which is responsible for corn mutants characterized by lowlevels of phytic acid. See Raboy et al., (2000).

A number of genes are known that may be used to alter carbohydratemetabolism. For example, plants may be transformed with a gene codingfor an enzyme that alters the branching pattern of starch. See Shirozaet al., (1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al., (1992)(production of transgenic plants that express Bacillus lichenifonnisα-amylase), U.S. Pat. No. 6,166,292 (low raffinose), Elliot et al.,(1993) (nucleotide sequences of tomato invertase genes), Sergaard etal., (1993) (site-directed mutagenesis of barley α-amylase gene), Fisheret al., (1993) (maize endosperm starch branching enzyme II), and U.S.Pat. Nos. 6,538,181; 6,538,179; 6,538,178; 5,750,876 and U.S. Pat. No.6,476,295 (starch content). The Z10 gene encoding a 10 kD zein storageprotein from maize may also be used to alter the quantities of 10 kDZein in the cells relative to other components (Kirihara et al., 1988).

Additional transgenes for use according to the invention include, butare not limited to, transgenes conferring increased intrinsic yield(e.g., eIF5A, deoxyhypusine synthase, serine carboxypeptidase, 2,4-Ddioxygenase), increased nutrient use efficiency, such as nitrogen useefficiency, increased cold tolerance, increased stress resistance andincreased drought tolerance (e.g., cspB, transcription factors).

II. ANTISENSE AND RNAI CONSTRUCTS

A transgene for use according to the invention may also comprise anantisense or RNAi coding sequence. Antisense methodology takes advantageof the fact that nucleic acids tend to pair with “complementary”sequences. By complementary, it is meant that polynucleotides are thosewhich are capable of base-pairing according to the standard Watson-Crickcomplementarity rules. That is, the larger purines will base pair withthe smaller pyrimidines to form combinations of guanine paired withcytosine (G:C) and adenine paired with either thymine (A:T) in the caseof DNA, or adenine paired with uracil (A:U) in the case of RNA.Inclusion of less common bases such as inosine, 5-methylcytosine,6-methyladenine, hypoxanthine and others in hybridizing sequences doesnot interfere with pairing.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. It is contemplated that the most effective antisense constructswill include regions complementary to intron/exon splice junctions.Thus, it is proposed that a preferred embodiment includes an antisenseconstruct with complementarity to regions within 50-200 bases of anintron-exon splice junction. It has been observed that some exonsequences can be included in the construct without seriously affectingthe target selectivity thereof. The amount of exonic material includedwill vary depending on the particular exon and intron sequences used.One can readily test whether too much exon DNA is included simply bytesting the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

As stated above, “complementary” or “antisense” means polynucleotidesequences that are substantially complementary over their entire lengthand have very few base mismatches. For example, sequences of fifteenbases in length may be termed complementary when they have complementarynucleotides at thirteen or fourteen positions. Naturally, sequenceswhich are completely complementary will be sequences which are entirelycomplementary throughout their entire length and have no basemismatches. Other sequences with lower degrees of homology also arecontemplated. For example, an antisense construct which has limitedregions of high homology, but also contains a non-homologous region(e.g., ribozyme; see above) could be designed. These molecules, thoughhaving less than 50% homology, would bind to target sequences underappropriate conditions.

It may be advantageous to combine portions of genomic DNA with cDNA orsynthetic sequences to generate specific constructs. For example, wherean intron is desired in the ultimate construct, a genomic clone willneed to be used. The cDNA or a synthesized polynucleotide may providemore convenient restriction sites for the remaining portion of theconstruct and, therefore, would be used for the rest of the sequence.

RNA interference (RNAi) is a process utilizing endogenous cellularpathways whereby a double stranded RNA (dsRNA) specific target generesults in the degradation of the mRNA of interest. In recent years,RNAi has been used to perform gene “knockdown” in a number of speciesand experimental systems, from the nematode C. elegans, to plants, toinsect embryos and cells in tissue culture (Fire et al., 1998; Martinezet al., 2002; McManus and Sharp, 2002). RNAi works through an endogenouspathway including the Dicer protein complex that generates˜21-nucleotide small interfering RNAs (siRNAs) from the original dsRNAand the RNA-induced silencing complex (RISC) that uses siRNA guides torecognize and degrade the corresponding mRNAs. Only transcriptscomplementary to the siRNA are cleaved and degraded, and thus theknock-down of mRNA expression is usually sequence specific. One of skillin the art would routinely be able to identify portions of, forinstance, an insect gene sequence, as targets for RNAi-mediated genesuppression to mediate morbidity and/or mortality in pest of interest.

III. TISSUE CULTURES

Tissue cultures may be used in certain transformation techniques for thepreparation of cells for transformation and for the regeneration ofplants therefrom. Maintenance of tissue cultures requires use of mediaand controlled environments. “Media” refers to the numerous nutrientmixtures that are used to grow cells in vitro, that is, outside of theintact living organism. The medium usually is a suspension of variouscategories of ingredients (salts, amino acids, growth regulators,sugars, buffers) that are required for growth of most cell types.However, each specific cell type requires a specific range of ingredientproportions for growth, and an even more specific range of formulas foroptimum growth. Rate of cell growth also will vary among culturesinitiated with the array of media that permit growth of that cell type.

Nutrient media is prepared as a liquid, but this may be solidified byadding the liquid to materials capable of providing a solid support.Agar is most commonly used for this purpose. Bacto™ agar (Difco-BD,Franklin Lakes, N.J.), Hazleton agar (Hazleton, Lenexa, Kans., USA),Gelrite® (Sigma, St. Louis, Mo.), PHYTAGEL (Sigma-Aldrich, St. Louis,Mo.), and GELGRO (ICN-MP Biochemicals, Irvine, Calif., USA) are specifictypes of solid support that are suitable for growth of plant cells intissue culture.

Some cell types will grow and divide either in liquid suspension or onsolid media. As disclosed herein, plant cells will grow in suspension oron solid medium, but regeneration of plants from suspension culturestypically requires transfer from liquid to solid media at some point indevelopment. The type and extent of differentiation of cells in culturewill be affected not only by the type of media used and by theenvironment, for example, pH, but also by whether media is solid orliquid.

Tissue that can be grown in a culture includes meristem cells, callus,immature embryos, hairy root cultures, and gametic cells such asmicrospores, pollen, sperm and egg cells. Callus may be initiated fromtissue sources including, but not limited to, immature embryos, seedlingapical meristems, root, leaf, microspores and the like. Those cellswhich are capable of proliferating as callus also are candidaterecipient cells for genetic transformation.

Somatic cells are of various types. Embryogenic cells are one example ofsomatic cells which may be induced to regenerate a plant through embryoformation. Non-embryogenic cells are those which typically will notrespond in such a fashion. Certain techniques may be used that enrichrecipient cells within a cell population, for example by manualselection and culture of friable, embryogenic tissue. Manual selectiontechniques which can be employed to select target cells may include,e.g., assessing cell morphology and differentiation, or may use variousphysical or biological means. Cryopreservation also is a possible methodof selecting for recipient cells.

Where employed, cultured cells may be grown either on solid supports orin the form of liquid suspensions. In either instance, nutrients may beprovided to the cells in the form of media, and environmental conditionscontrolled. There are many types of tissue culture media comprised ofvarious amino acids, salts, sugars, growth regulators and vitamins. Mostof the media employed in the practice of the invention will have somesimilar components, but may differ in the composition and proportions oftheir ingredients depending on the particular application envisioned.For example, various cell types usually grow in more than one type ofmedia, but will exhibit different growth rates and differentmorphologies, depending on the growth media. In some media, cellssurvive but do not divide. Various types of media suitable for cultureof plant cells previously have been described. Examples of these mediainclude, but are not limited to, the N6 medium described by Chu et al.,(1975) and MS media (Murashige and Skoog, 1962).

IV. METHODS FOR GENETIC TRANSFORMATION

Suitable methods for transformation of plant or other cells for use withthe current invention are believed to include virtually any method bywhich DNA can be introduced into a cell, such as by direct delivery ofDNA such as by PEG-mediated transformation of protoplasts (Omirulleh etal., 1993), by desiccation/inhibition-mediated DNA uptake (Potrykus etal., 1985), by electroporation (U.S. Pat. No. 5,384,253, specificallyincorporated herein by reference in its entirety), by agitation withsilicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. No. 5,302,523,specifically incorporated herein by reference in its entirety; and U.S.Pat. No. 5,464,765, specifically incorporated herein by reference in itsentirety), by Agrobacterium-mediated transformation (U.S. Pat. No.5,591,616 and U.S. Pat. No. 5,563,055; both specifically incorporatedherein by reference) and by acceleration of DNA coated particles (U.S.Pat. No. 5,550,318; U.S. Pat. No. 5,538,877; and U.S. Pat. No.5,538,880; each specifically incorporated herein by reference in itsentirety), etc. Through the application of techniques such as these, thecells of virtually any plant species may be stably transformed, andthese cells developed into transgenic plants.

A. Agrobacterium-Mediated Transformation

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described by Fraley etal., (1985), Rogers et al., (1987) and U.S. Pat. No. 5,563,055,specifically incorporated herein by reference in its entirety.

Agrobacterium-mediated transformation is most efficient indicotyledonous plants and is the preferable method for transformation ofdicots, including Arabidopsis, tobacco, tomato, alfalfa and potato.Indeed, while Agrobacterium-mediated transformation has been routinelyused with dicotyledonous plants for a number of years, it has onlyrecently become applicable to monocotyledonous plants. Advances inAgrobacterium-mediated transformation techniques have now made thetechnique applicable to nearly all monocotyledonous plants. For example,Agrobacterium-mediated transformation techniques have now been appliedto rice (Hiei et al., 1997; U.S. Pat. No. 5,591,616), wheat (McCormac etal., 1998), barley (Tingay et al., 1997; McCormac et al., 1998), alfalfa(e.g., Thomas et al., 1990; McKersie et al., 1993) and maize (Ishida etal., 1996).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described(Rogers et al., 1987) have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes. Inaddition, Agrobacterium containing both armed and disarmed Ti genes canbe used for the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

B. Electroporation

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wounding in acontrolled manner. Examples of some species which have been transformedby electroporation of intact cells include maize (U.S. Pat. No.5,384,253; Rhodes et al., 1995; D'Halluin et al., 1992), wheat (Zhou etal., 1993), tomato (Hou and Lin, 1996), soybean (Christou et al., 1987)and tobacco (Lee et al., 1989).

One also may employ protoplasts for electroporation transformation ofplants (Bates, 1994; Lazzeri, 1995). For example, the generation oftransgenic soybean plants by electroporation of cotyledon-derivedprotoplasts is described by Dhir and Widholm in Intl. Patent Appl. Publ.No. WO 9217598 (specifically incorporated herein by reference). Otherexamples of species for which protoplast transformation has beendescribed include barley (Lazerri, 1995), sorghum (Battraw et al.,1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994) andtomato (Tsukada, 1989).

C. Microprojectile Bombardment

Another method for delivering transforming DNA segments to plant cellsin accordance with the invention is microprojectile bombardment (U.S.Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No. 5,610,042;and PCT Application WO 94/09699; each of which is specificallyincorporated herein by reference in its entirety). In this method,particles may be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, platinum, and preferably, gold. It is contemplated that insome instances DNA precipitation onto metal particles would not benecessary for DNA delivery to a recipient cell using microprojectilebombardment. However, it is contemplated that particles may contain DNArather than be coated with DNA. Hence, it is proposed that DNA-coatedparticles may increase the level of DNA delivery via particlebombardment but are not, in and of themselves, necessary.

For the bombardment, cells in suspension are concentrated on filters orsolid culture medium. Alternatively, immature embryos or other targetcells may be arranged on solid culture medium. The cells to be bombardedare positioned at an appropriate distance below the macroprojectilestopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics® Particle Delivery System(Dupont), which can be used to propel particles coated with DNA or cellsthrough a screen, such as a stainless steel or nylon screen (e.g., NYTEXscreen; Sefar America, Depew, N.Y. USA), onto a filter surface coveredwith plant cells cultured in suspension. The screen disperses theparticles so that they are not delivered to the recipient cells in largeaggregates. Microprojectile bombardment techniques are widelyapplicable, and may be used to transform virtually any plant species.Examples of species for which have been transformed by microprojectilebombardment include monocot species such as maize (PCT Application WO95/06128), barley (Ritala et al., 1994), wheat (U.S. Pat. No.5,563,055), and sorghum (Casa et al., 1993); as well as a number ofdicots including tobacco (Tomes et al., 1990; Buising and Benbow, 1994),soybean (U.S. Pat. No. 5,322,783), sunflower (Knittel et al., 1994),peanut (Singsit et al., 1997), cotton (McCabe and Martinell, 1993),tomato (VanEck et al., 1995), and legumes in general (U.S. Pat. No.5,563,055, specifically incorporated herein by reference in itsentirety).

D. Other Transformation Methods

Transformation of protoplasts can be achieved using methods based oncalcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Lorz et al., 1985; Omirulleh et al., 1993; Frommet al., 1986; Uchimiya et al., 1986; Callis et al., 1987; Marcotte etal., 1988).

Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Illustrative methods for the regeneration of plants from protoplastshave been described (Toriyama et al., 1986; Yamada et al., 1986;Abdullah et al., 1986; Omirulleh et al., 1993 and U.S. Pat. No.5,508,184). Examples of the use of direct uptake transformation ofprotoplasts include transformation of rice (Ghosh-Biswas et al., 1994),sorghum (Battraw and Hall, 1991), barley (Lazerri, 1995), oat (Zheng andEdwards, 1990) and maize (Omirulleh et al., 1993).

To transform plant strains that cannot be successfully regenerated fromprotoplasts, other ways to introduce DNA into intact cells or tissuescan be utilized. For example, regeneration of cereals from immatureembryos or explants can be effected as described (Vasil, 1989). Also,silicon carbide fiber-mediated transformation may be used with orwithout protoplasting (Kaeppler, 1990; Kaeppler et al., 1992; U.S. Pat.No. 5,563,055). Transformation with this technique is accomplished byagitating silicon carbide fibers together with cells in a DNA solution.DNA passively enters as the cells are punctured. This technique has beenused successfully with, for example, the monocot cereals maize (PCTApplication WO 95/06128; (Thompson, 1995) and rice (Nagatani, 1997).

V. PRODUCTION AND CHARACTERIZATION OF STABLY TRANSFORMED PLANTS

After effecting delivery of exogenous DNA to recipient cells, the nextsteps generally concern identifying the transformed cells for furtherculturing and plant regeneration. In order to improve the ability toidentify transformants, one may desire to employ a selectable orscreenable marker gene with a transformation vector prepared inaccordance with the invention. In this case, one would then generallyassay the potentially transformed cell population by exposing the cellsto a selective agent or agents, or one would screen the cells for thedesired marker gene trait.

A. Selection

It is believed that DNA is introduced into only a small percentage oftarget cells in any one experiment. In order to provide an efficientsystem for identification of those cells receiving DNA and integratingit into their genomes one may employ a means for selecting those cellsthat are stably transformed. One exemplary embodiment of such a methodis to introduce into the host cell, a marker gene which confersresistance to some normally inhibitory agent, such as an antibiotic orherbicide. Examples of antibiotics which may be used include theaminoglycoside antibiotics neomycin, kanamycin and paromomycin, or theantibiotic hygromycin. Resistance to the aminoglycoside antibiotics isconferred by aminoglycoside phosphostransferase enzymes such as neomycinphosphotransferase II (NPT II) or NPT I, whereas resistance tohygromycin is conferred by hygromycin phosphotransferase.

Potentially transformed cells then are exposed to the selective agent.In the population of surviving cells will be those cells where,generally, the resistance-conferring gene has been integrated andexpressed at sufficient levels to permit cell survival. Cells may betested further to confirm stable integration of the exogenous DNA.

One herbicide which constitutes a desirable selection agent is the broadspectrum herbicide bialaphos. Bialaphos is a tripeptide antibioticproduced by Streptomyces hygroscopicus and is composed ofphosphinothricin (PPT), an analogue of L-glutamic acid, and twoL-alanine residues. Upon removal of the L-alanine residues byintracellular peptidases, the PPT is released and is a potent inhibitorof glutamine synthetase (GS), a pivotal enzyme involved in ammoniaassimilation and nitrogen metabolism (Ogawa et al., 1973). SyntheticPPT, the active ingredient in the herbicide Liberty™ also is effectiveas a selection agent. Inhibition of GS in plants by PPT causes the rapidaccumulation of ammonia and death of the plant cells.

The organism producing bialaphos and other species of the genusStreptomyces also synthesizes an enzyme phosphinothricin acetyltransferase (PAT) which is encoded by the bar gene in Streptomyceshygroscopicus and the pat gene in Streptomyces viridochromogenes. Theuse of the herbicide tolerance gene encoding phosphinothricin acetyltransferase (PAT) is referred to in DE 3642 829 A, wherein the gene isisolated from Streptomyces viridochromogenes. In the bacterial sourceorganism, this enzyme acetylates the free amino group of PPT preventingauto-toxicity (Thompson et al., 1987). The bar gene has been cloned(Murakami et al., 1986; Thompson et al., 1987) and expressed intransgenic tobacco, tomato, potato (De Block et al., 1987) Brassica (DeBlock et al., 1989) and maize (U.S. Pat. No. 5,550,318). In previousreports, some transgenic plants which expressed the resistance gene werecompletely resistant to commercial formulations of PPT and bialaphos ingreenhouses.

Another example of a herbicide which is useful for selection oftransformed cell lines in the practice of the invention is the broadspectrum herbicide glyphosate. Glyphosate inhibits the action of theenzyme EPSPS which is active in the aromatic amino acid biosyntheticpathway. Inhibition of this enzyme leads to starvation for the aminoacids phenylalanine, tyrosine, and tryptophan and secondary metabolitesderived thereof. U.S. Pat. No. 4,535,060 describes the isolation ofEPSPS mutations which confer glyphosate resistance on the Salmonellatyphimurium gene for EPSPS, aroA. The EPSPS gene was cloned from Zeamays and mutations similar to those found in a glyphosate resistant aroAgene were introduced in vitro. Mutant genes encoding glyphosateresistant EPSPS enzymes are described in, for example, InternationalPatent WO 97/4103. The best characterized mutant EPSPS gene conferringglyphosate resistance comprises amino acid changes at residues 102 and106, although it is anticipated that other mutations will also be useful(PCT/WO97/4103).

To use the bar-bialaphos or the EPSPS-glyphosate selective system,transformed tissue is cultured for 0-28 days on nonselective medium andsubsequently transferred to medium containing from 1-3 mg/l bialaphos or1-3 mM glyphosate as appropriate. While ranges of 1-3 mg/l bialaphos or1-3 mM glyphosate will typically be preferred, it is proposed thatranges of 0.1-50 mg/l bialaphos or 0.1-50 mM glyphosate will findutility.

It further is contemplated that the herbicide DALAPON,2,2-dichloropropionic acid, may be useful for identification oftransformed cells. The enzyme 2,2-dichloropropionic acid dehalogenase(deh) inactivates the herbicidal activity of 2,2-dichloropropionic acidand therefore confers herbicidal resistance on cells or plantsexpressing a gene encoding the dehalogenase enzyme (Buchanan-Wollastonet al., 1992; U.S. Pat. No. 5,508,468).

Alternatively, a gene encoding anthranilate synthase, which confersresistance to certain amino acid analogs, e.g., 5-methyltryptophan or6-methyl anthranilate, may be useful as a selectable marker gene. Theuse of an anthranilate synthase gene as a selectable marker wasdescribed in U.S. Pat. No. 5,508,468.

An example of a screenable marker trait is the enzyme luciferase. In thepresence of the substrate luciferin, cells expressing luciferase emitlight which can be detected on photographic or x-ray film, in aluminometer (or liquid scintillation counter), by devices that enhancenight vision, or by a highly light sensitive video camera, such as aphoton counting camera. These assays are nondestructive and transformedcells may be cultured further following identification. The photoncounting camera is especially valuable as it allows one to identifyspecific cells or groups of cells which are expressing luciferase andmanipulate those in real time. Another screenable marker which may beused in a similar fashion is the gene coding for green fluorescentprotein.

It further is contemplated that combinations of screenable andselectable markers will be useful for identification of transformedcells. In some cell or tissue types a selection agent, such as bialaphosor glyphosate, may either not provide enough killing activity to clearlyrecognize transformed cells or may cause substantial nonselectiveinhibition of transformants and nontransformants alike, thus causing theselection technique to not be effective. It is proposed that selectionwith a growth inhibiting compound, such as bialaphos or glyphosate atconcentrations below those that cause 100% inhibition followed byscreening of growing tissue for expression of a screenable marker genesuch as luciferase would allow one to recover transformants from cell ortissue types that are not amenable to selection alone. It is proposedthat combinations of selection and screening may enable one to identifytransformants in a wider variety of cell and tissue types. This may beefficiently achieved using a gene fusion between a selectable markergene and a screenable marker gene, for example, between an NPTII geneand a GFP gene.

B. Regeneration and Seed Production

Cells that survive the exposure to the selective agent, or cells thathave been scored positive in a screening assay, may be cultured in mediathat supports regeneration of plants. In an exemplary embodiment, MS andN6 media may be modified by including further substances such as growthregulators. One such growth regulator is dicamba or 2,4-D. However,other growth regulators may be employed, including NAA, NAA+2,4-D orpicloram. Media improvement in these and like ways has been found tofacilitate the growth of cells at specific developmental stages. Tissuemay be maintained on a basic media with growth regulators untilsufficient tissue is available to begin plant regeneration efforts, orfollowing repeated rounds of manual selection, until the morphology ofthe tissue is suitable for regeneration, at least 2 wk, then transferredto media conducive to maturation of embryoids. Cultures are transferredevery 2 wk on this medium. Shoot development will signal the time totransfer to medium lacking growth regulators.

The transformed cells, identified by selection or screening and culturedin an appropriate medium that supports regeneration, will then beallowed to mature into plants. Developing plantlets are transferred tosoiless plant growth mix, and hardened, e.g., in an environmentallycontrolled chamber, for example, at about 85% relative humidity, 600 ppmCO₂, and 25-250 microeinsteins m⁻² s⁻¹ of light. Plants are preferablymatured either in a growth chamber or greenhouse. Plants can beregenerated from about 6 wk to 10 months after a transformant isidentified, depending on the initial tissue. During regeneration, cellsare grown on solid media in tissue culture vessels. Illustrativeembodiments of such vessels are petri dishes and Plantcon™ containers(MP-ICN Biomedicals, Solon, Ohio, USA). Regenerating plants arepreferably grown at about 19 to 28° C. After the regenerating plantshave reached the stage of shoot and root development, they may betransferred to a greenhouse for further growth and testing.

Seeds on transformed plants may occasionally require embryo rescue dueto cessation of seed development and premature senescence of plants. Torescue developing embryos, they are excised from surface-disinfectedseeds 10-20 days post-pollination and cultured. An embodiment of mediaused for culture at this stage comprises MS salts, 2% sucrose, and 5.5g/l agarose. In embryo rescue, large embryos (defined as greater than 3mm in length) are germinated directly on an appropriate media. Embryossmaller than that may be cultured for 1 wk on media containing the aboveingredients along with 10⁻⁵ M abscisic acid and then transferred togrowth regulator-free medium for germination.

C. Characterization

To confirm the presence of the exogenous DNA or “transgene(s)” in theregenerating plants, a variety of assays may be performed. Such assaysinclude, for example, “molecular biological” assays, such as Southernand northern blotting and PCR; “biochemical” assays, such as detectingthe presence of a protein product, e.g., by immunological means (ELISAsand Western blots) or by enzymatic function; plant part assays, such asleaf or root assays; and also, by analyzing the phenotype of the wholeregenerated plant.

D. DNA Integration, RNA Expression and Inheritance

Genomic DNA may be isolated from cell lines or any plant parts todetermine the presence of the exogenous gene through the use oftechniques well known to those skilled in the art. Note, that intactsequences will not always be present, presumably due to rearrangement ordeletion of sequences in the cell. The presence of DNA elementsintroduced through the methods of this invention may be determined, forexample, by polymerase chain reaction (PCR). Using this technique,discreet fragments of DNA are amplified and detected by gelelectrophoresis. This type of analysis permits one to determine whethera gene is present in a stable transformant, but does not proveintegration of the introduced gene into the host cell genome. It istypically the case, however, that DNA has been integrated into thegenome of all transformants that demonstrate the presence of the genethrough PCR analysis. In addition, it is not typically possible usingPCR™ techniques to determine whether transformants have exogenous genesintroduced into different sites in the genome, i.e., whethertransformants are of independent origin. It is contemplated that usingPCR techniques it would be possible to clone fragments of the hostgenomic DNA adjacent to an introduced gene.

Positive proof of DNA integration into the host genome and theindependent identities of transformants may be determined using thetechnique of Southern hybridization. Using this technique specific DNAsequences that were introduced into the host genome and flanking hostDNA sequences can be identified. Hence the Southern hybridizationpattern of a given transformant serves as an identifying characteristicof that transformant. In addition it is possible through Southernhybridization to demonstrate the presence of introduced genes in highmolecular weight DNA, i.e., confirm that the introduced gene has beenintegrated into the host cell genome. The technique of Southernhybridization provides information that is obtained using PCR, e.g., thepresence of a gene, but also demonstrates integration into the genomeand characterizes each individual transformant.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA will only be expressed in particular cells ortissue types and hence it will be necessary to prepare RNA for analysisfrom these tissues. PCR techniques also may be used for detection andquantitation of RNA produced from introduced genes. In this applicationof PCR it is first necessary to reverse transcribe RNA into DNA, usingenzymes such as reverse transcriptase, and then through the use ofconventional PCR techniques amplify the DNA. In most instances PCRtechniques, while useful, will not demonstrate integrity of the RNAproduct. Further information about the nature of the RNA product may beobtained by northern blotting. This technique will demonstrate thepresence of an RNA species and give information about the integrity ofthat RNA. The presence or absence of an RNA species also can bedetermined using dot or slot blot northern hybridizations. Thesetechniques are modifications of northern blotting and will onlydemonstrate the presence or absence of an RNA species.

E. Gene Expression

While Southern blotting and PCR may be used to detect the gene(s) inquestion, they do not provide information as to whether thecorresponding protein is being expressed. Expression may be evaluated bydetermining expression via transcript-profiling techniques such as byuse of a microarray, and by specifically identifying the proteinproducts of the introduced genes or evaluating the phenotypic changesbrought about by their expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focusing, or by chromatographictechniques such as ion exchange or gel exclusion chromatography. Theunique structures of individual proteins offer opportunities for use ofspecific antibodies to detect their presence in formats such as an ELISAassay. Combinations of approaches may be employed with even greaterspecificity such as western blotting in which antibodies are used tolocate individual gene products that have been separated byelectrophoretic techniques. Additional techniques may be employed toabsolutely confirm the identity of the product of interest such asevaluation by amino acid sequencing following purification. Althoughthese are among the most commonly employed, other procedures may beadditionally used.

Assay procedures also may be used to identify the expression of proteinsby their functionality, especially the ability of enzymes to catalyzespecific chemical reactions involving specific substrates and products.These reactions may be followed by providing and quantifying the loss ofsubstrates or the generation of products of the reactions by physical orchemical procedures. Examples are as varied as the enzyme to be analyzedand may include assays for PAT enzymatic activity by followingproduction of radiolabeled acetylated phosphinothricin fromphosphinothricin and ¹⁴C-acetyl CoA or for anthranilate synthaseactivity by following loss of fluorescence of anthranilate, to name two.

Very frequently the expression of a gene product is determined byevaluating the phenotypic results of its expression. These assays alsomay take many forms including but not limited to analyzing changes inthe chemical composition, morphology, or physiological properties of theplant. Chemical composition may be altered by expression of genesencoding enzymes or storage proteins which change amino acid compositionand may be detected by amino acid analysis, or by enzymes which changestarch quantity which may be analyzed by near infrared reflectancespectrometry. Morphological changes may include greater stature orthicker stalks. Most often changes in response of plants or plant partsto imposed treatments are evaluated under carefully controlledconditions termed bioassays.

VI. BREEDING PLANTS OF THE INVENTION

In addition to direct transformation of a particular plant genotype witha construct prepared according to the current invention, transgenicplants may be made by crossing a plant having a selected DNA of theinvention to a second plant lacking the construct. For example, aselected CT biosynthesis gene can be introduced into a particular plantvariety by crossing, without the need for ever directly transforming aplant of that given variety. Therefore, the current invention not onlyencompasses a plant directly transformed or regenerated from cells whichhave been transformed in accordance with the current invention, but alsothe progeny of such plants. As used herein the term “progeny” denotesthe offspring of any generation of a parent plant prepared in accordancewith the invention, wherein the progeny comprises a selected DNAconstruct prepared in accordance with the invention. “Crossing” a plantto provide a plant line having one or more added transgenes relative toa starting plant line, as disclosed herein, is defined as the techniquesthat result in a transgene of the invention being introduced into aplant line by crossing a starting line with a donor plant line thatcomprises a transgene of the invention. To achieve this one could, forexample, perform the following steps:

(a) plant seeds of the first (starting line) and second (donor plantline that comprises a transgene of the invention) parent plants;

(b) grow the seeds of the first and second parent plants into plantsthat bear flowers;

(c) pollinate a flower from the first parent plant with pollen from thesecond parent plant; and

(d) harvest seeds produced on the parent plant bearing the fertilizedflower.

Backcrossing is herein defined as the process including the steps of:

(a) crossing a plant of a first genotype containing a desired gene, DNAsequence or element to a plant of a second genotype lacking the desiredgene, DNA sequence or element;

(b) selecting one or more progeny plant containing the desired gene, DNAsequence or element;

(c) crossing the progeny plant to a plant of the second genotype; and

(d) repeating steps (b) and (c) for the purpose of transferring adesired DNA sequence from a plant of a first genotype to a plant of asecond genotype.

Introgression of a DNA element into a plant genotype is defined as theresult of the process of backcross conversion. A plant genotype intowhich a DNA sequence has been introgressed may be referred to as abackcross converted genotype, line, inbred, or hybrid. Similarly a plantgenotype lacking the desired DNA sequence may be referred to as anunconverted genotype, line, inbred, or hybrid.

VII. DEFINITIONS

Expression: The combination of intracellular processes, includingtranscription. In the case of a functional RNA sequence, such as anantisense RNA, siRNA or miroc RNA, expression may involve transcriptionand processing of the functional RNA. In the case of a polypeptidecoding sequence expression includes trancription and translation toproduce a polypeptide.

Expression Cassette: A transgene operably linked to nucleic acidsequences that control expression of the transgene in a cell. Expressioncontrol, sequences include but are not limited to promoters, enhancers,introns, termininators and internal ribosome entry sites.

Genetic Transformation: A process of introducing a DNA sequence orconstruct (e.g., a vector or expression cassette) into a cell orprotoplast in which that exogenous DNA is incorporated into a chromosomeor is capable of autonomous replication.

Heterologous: A sequence which is not normally present in a given hostgenome in the genetic context in which the sequence is currently foundIn this respect, the sequence may be native to the host genome, but berearranged with respect to other genetic sequences within the hostsequence. For example, a regulatory sequence may be heterologous in thatit is linked to a different coding sequence relative to the nativeregulatory sequence.

Obtaining: When used in conjunction with a transgenic plant cell ortransgenic plant, obtaining means either transforming a non-transgenicplant cell or plant to create the transgenic plant cell or plant, orplanting transgenic plant seed to produce the transgenic plant cell orplant. Such a transgenic plant seed may be from an R₀ transgenic plantor may be from a progeny of any generation thereof that inherits a giventransgenic sequence from a starting transgenic parent plant.

Promoter: A recognition site on a DNA sequence or group of DNA sequencesthat provides an expression control element for a structural gene and towhich RNA polymerase specifically binds and initiates RNA synthesis(transcription) of that gene.

R₀ transgenic plant: A plant that has been genetically transformed orhas been regenerated from a plant cell or cells that have beengenetically transformed.

Regeneration: The process of growing a plant from a plant cell (e.g.,plant protoplast, callus or explant).

Selected DNA: A DNA segment which one desires to introduce into a plantgenome by genetic transformation.

Transformation construct: A chimeric DNA molecule which is designed forintroduction into a host genome by genetic transformation. Preferredtransformation constructs will comprise all of the genetic elementsnecessary to direct the expression of one or more exogenous genes. Inparticular embodiments of the instant invention, it may be desirable tointroduce a transformation construct into a host cell in the form of anexpression cassette.

Transformed cell: A cell the DNA complement of which has been altered bythe introduction of an exogenous DNA molecule into that cell.

Transgene: A segment of DNA which has been incorporated into a hostgenome or is capable of autonomous replication in a host cell and iscapable of causing the expression of one or more RNAs and/orpolypeptides. Exemplary transgenes will provide the host cell, or plantsregenerated therefrom, with a novel phenotype relative to thecorresponding non-transformed cell or plant. Transgenes may be directlyintroduced into a plant by genetic transformation, or may be inheritedfrom a plant of any previous generation which was transformed with theDNA segment.

Transgenic event: A transgenic “event” is produced by transformation ofthe genome of a plant with a heterologous DNA construct, including aconstruct that comprises a plurality of transgenes in accordance withthe invention. The term “event” refers to the original transformant andincludes any progeny that inherit the event, such as by sexualoutcrossing. Through standard plant breeding, it is understood that oneof skill in the art can introduce a given transformation event into anyother genetic background that is sexually compatible with a startingplant comprising the event.

Transgenic plant: A plant or progeny plant of any subsequent generationderived therefrom, wherein the DNA of the plant or progeny thereofcontains an introduced exogenous DNA segment not naturally present in anon-transgenic plant of the same strain. The transgenic plant mayadditionally contain sequences which are native to the plant beingtransformed, but wherein the “exogenous” gene has been altered in orderto alter the level or pattern of expression of the gene, for example, byuse of one or more heterologous regulatory or other elements.

Vector: A DNA molecule capable of replication in a host cell and/or towhich another DNA segment can be operatively linked so as to bring aboutreplication of the attached segment. A plasmid is an exemplary vector.

VIII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1 Construction of Vectors with Multiple Expression Cassettes

Vectors were constructed comprising either 6 or 10 expression cassettes(and 6 or 10 distinct genes). Table 1 illustrates the traits conferredby each of the transgenes in the multi-transgene vectors. Hrb1, Hrb2 andHrb3; represent herbicide tolerance genes, Dr1 and Dr2 represent droughtresistance genes; Ins1, Ins2, Ins3 and Ins4 represent insect resistancegenes; and Sel represents a selectable marker gene. Also shown in Table1 are the expression control sequences, promoter, intron and terminatorthat were included in each vector. Table 2 shows the number of timeseach promoter, intron and terminator sequence were repeated in the 10gene construct. For construction of the multi-transgene vectorsindividual blocks of expression cassette sequences were cloned into aparent vector and the GATEWAY™ recombinase system was used to generatethe fully formed multiple gene vector. The GATEWAY™ recombinationreaction was used to directly transform Agrobacterium. Bacterialcolonies were screened to identify one which contained the fully formed10 gene insert. A schematic representation of the binary T-DNA vectorused for recombination and the final T-DNA vector including the ˜32 KB,10 gene insertion is shown as FIG. 1.

TABLE 1 Expression cassettes on the 10 gene vector Cassette Trait(promoter/intron/gene/terminator) Herbicide Tolerance*ePCISV/Ract1/Hrb1/Hsp17 Herbicide Tolerance* TubA/TubA/Hrb2/TubAHerbicide Tolerance* eFMV/Sus1/Hrb3/Ara5 Insect Resistance(below-ground) e35S/Hsp70/Ins1/35S Insect Resistance (below-ground)e35S/Ract1/Ins2/Hsp17 Insect Resistance (above-ground)e35S/Ract1/Ins3/Hsp17 Insect Resistance (above-ground)FMV/Hsp70/Ins4/nos Drought Tolerance* Ract1/Ract1/Dr1/Tr7 DroughtTolerance* TubA/TubA/Dr2/TubA Selectable Marker* 35S/Sel/nos *=expression cassettes in the 6 gene vector.

TABLE 2 Repetition of control sequences in the 10 gene vector Controlelement Number of repeats 35S promoter 4 Os.Act intron 4 Hsp17terminator 3 Hsp70 intron 2 Nos terminator 2 FMV promoter 2 TubApromoter 2 TubA intron 2 TubA terminator 2 Unique elements 6

Example 2 Transformation of Plant Cells

Agrobacterium comprising the 10 and 6 gene vectors were used fortransformation of maize immature embryos. Transformation efficiency andquality (percent single copy, backbone free sequence) was assess for the6 and 10 gene vectors and compared to a control, 2 gene, transformation.Results of these analyses are shown in Table 3. Most transgenic eventsexpressed all transgenes.

TABLE 3 Transformation Efficiency T-DNA % single copy Stack Size (kb)Explants Events TF(%) backbone free 2 gene 6.6 440 79 18.0 65.8 6 gene17 440 69 15.7 39.1 10 gene  32 440 20 4.5 55.0

Results of the transformation analysis demonstrated that transformationefficiency (TF) decreases as the number of genes in the transformationvector increased. Surprisingly, however, the 10 gene vector demonstratedenhanced transformation quality relative to the 6 gene vector andgenerated a significantly greater portion of single copy, backbone freeinserts.

Example 3 Characterization of Transformed Plant Cells

Transgenic events comprising the 10 gene vector were next subjected toSouthern blot analysis to determine whether the transformation eventscomprised the intact 10 gene insertion. Results of the analysis from 9events are shown in FIG. 2. Results of the analysis demonstrate thatmost events contained one, intact T-DNA insert. These results werefurther confirmed by FISH analysis of the three events shown in FIG. 3.

The 10 gene transformation events were next characterized for expressionof the various transgenes. Table 4 shows the relative RNA expressionfrom each of the transgenes compared to control expression vectors. Inthe case of Dr1 and Dr2, expression was compared to expression in a linecomprising a transformation event of two transgenes (Dr1 and Dr2).Likewise for Ins1 and Ins2 expression was compared to expression in aline comprising a transformation event of two transgenes (Ins1 andIns2). In the case of Hrb1, Hrb2 and Hrb3, expression was compared toexpression in a line comprising a transformation event of threetransgenes (Hrb1, Hrb2 and Hrb3). The far right two columns showabsolute RNA levels as no control levels were obtained.

RNA expression results for individual transformants were also plotted ona graph as shown in FIG. 4. In each case the horizontal line indicatesthe level of expression from plants transformed with control vectorsdescribed above that did not comprise the 10 gene stack.

TABLE 4 Relative RNA expression from 10 gene transformation eventsPercent RNA Expression Compared to Control Relative Expression Event NoDr2 Dr1 Ins1 Ins2 Hrb3 Hrb2 Hrb1 Nptll Ins4 ZM_1 17 618 393 198 190 107148 286 1271991 865519 ZM_2 14 632 335 151 141 91 134 339 12374455554635 ZM_3 10 237 176 124 110 141 137 306 497080 1354311 ZM_4 6 * * 6085 85 129 75 * 1034073 ZM_5 10 500 265 100 106 100 180 164 727638 771678ZM_6 7 962 237 101 311 447 10 211 1808399 619265 ZM_7 5 1080 319 120 112125 241 164 1642431 764004 ZM_8 6 392 220 54 90 20763 38 0 670331499139 * = Not tested.

Expression from the 10 gene transformation events was further analyzedto determine the relative protein expression levels. Results of theprotein expression analysis are shown in Table 5. In the case of Hrb1,Hrb2 and Hrb3, expression was compared to expression in a linecomprising a transformation event of three transgenes (Hrb1, Hrb2 andHrb3). For Ins1 expression was compared to expression in a linecomprising a transformation event of two transgenes (Ins1 and Ins2).

TABLE 5 Relative protein expression from 10 gene transformation eventsPercent Protein Compared Protein Percent Ins2 to Control (ppm/Fresh wt)compared to Event N Ins2 Hrb3 Hrb2 Hrb1 Ins4 Ins3 control* ZM_1 10 137146 86 84 11.76 0.43 43 ZM_2 10 179 200 82 81 42.07 0.60 56 ZM_3 10 226131 66 69 8.97 0.45 83 ZM_4 7 221 131 66 64 11.17 0.42 69 ZM_5 10 175130 77 77 10.42 0.47 55 ZM_6 7 581 404 6 70 7.22 0.38 182 ZM_7 5 328 15987 91 12.50 0.53 103 ZM_9 8 268 132 78 56 10.45 0.47 84 ZM_8 6 318 29530 ND 7.53 0.49 100 *Expression was compared to expression in a linecomprising a transformation event of a single Ins 2 transgene.

Example 4 Analysis of Traits Conferred by Transgenes in the 10 GeneTransformation Events

Studies were conducted to determine whether expression from the genes inthe 10 gene transformation events was sufficient to confer traits ofagronomic interest to transformed plants. In a first study, transformedplants were tested for their ability to destroy three types of aboveground pests. Results shown in FIG. 5 demonstrate that 10 genetransformed plants were able to destroy the three above-ground pests ata similar rate compared to plants expressing the same genes notcomprised in the 10 gene system. Leaf damage was also assessed in theplants and results shown in FIG. 6 demonstrate that the 10 genetransformation events exhibited similar or less leaf damage as comparedto plants expressing the same genes not comprised in the 10 gene system.In each case, “control” indicates results from a plant line transformedwith a two transgene vector (encoding the Ins3 and Ins4 transgenes).

Similar studies were undertaken to determine whether expression ofinsert resistance genes in the 10 gene transformation events were ableto protect roots from damage due to Coleoptera pests. The graph in FIG.7 demonstrated that genes expressed in the 10 gene transformation eventsprotected the plants at least as well or better than identical genesexpressed in plants that did not have the 10 gene stack. Control 1 is aplant line transformed with a two transgene vector (encoding the Ins1and Ins2 transgenes). Control 2 is a plant line transformed with asingle transgene vector (encoding the Ins2 transgene).

What is claimed is:
 1. A method for expressing at least 10 transgenes ina plant comprising: expressing the at least 10 transgenes in the plant,wherein the transgenes are arranged in tandem in a single locus of theplant, the at least 10 transgenes each being operably linked to apromoter sequence, wherein the at least 10 transgenes are transformedinto the single locus as a single transformation event with atransformation construct comprising the at least 10 transgenes, andwherein each of the at least 10 transgenes are expressed at levelssufficient to confer a selectable or screenable marker phenotype or atrait of agronomic interest to the plant.
 2. The method of claim 1,wherein expression of at least one of the at least 10 transgenes in saidplant is enhanced relative to expression of the same transgene in alocus of an otherwise isogenic plant having fewer than 10 transgenes. 3.The method of claim 1, wherein expression of at least two of the atleast 10 transgenes in said plant is enhanced relative to expression ofthe same transgenes in an otherwise isogenic plant having fewer than 10transgenes.
 4. The method of claim 1, wherein at least one of thetransgenes confers a trait of agronomic interest to the plant.
 5. Themethod of claim 4, wherein the trait of agronomic interest conferred bythe at least one transgene is selected from the group consisting ofherbicide tolerance, drought resistance, insect resistance, fungusresistance, virus resistance, bacteria resistance, male sterility, coldtolerance, salt tolerance, increased yield, enhanced oil composition,increased oil content, enhanced nutrient use efficiency and alteredamino acid content.
 6. The method of claim 1, wherein the single locuscomprises at least one transgene that confers herbicide tolerance and atleast one transgene that confers insect resistance.
 7. The method ofclaim 6, wherein the single locus comprises at least two transgenes thatconfer herbicide tolerance and at least two transgenes that conferinsect resistance.
 8. The method of claim 1, wherein the single locuscomprises at least one transgene that confers above-ground insectresistance and at least one transgene that confers below-ground insectresistance.
 9. The method of claim 1, wherein the plant is selected fromthe group consisting of wheat, maize, rye, rice, corn, oat, barley,turfgrass, sorghum, millet, sugarcane, tobacco, tomato, potato, soybean,cotton, canola, sunflower and alfalfa.
 10. The method of claim 1,wherein at least 10 of the transgenes in the single locus of the plantare each expressed at levels sufficient to confer a trait of agronomicinterest to the plant.
 11. The method of claim 1, wherein expression ofeach of the at least 10 transgenes in said plant is enhanced relative toexpression of the same transgenes in one or more otherwise isogenicplants each having fewer than 10 transgenes.
 12. The method of claim 1,wherein the at least 10 transgenes are transformed into the single locusvia Agrobacterium-mediated transformation.
 13. The method of claim 6,wherein said locus comprises at least one transgene that confers droughttolerance.
 14. The method of claim 9, wherein the plant is a corn plant.15. A method of making a transgenic plant comprising: transforming viaAgrobacterium-mediated transformation at least 10 transgenes into asingle locus of an explant, the transformation into the single locusoccurring as a single transformation event with a transformationconstruct comprising the at least 10 transgenes, wherein each of the atleast 10 transgenes is operatively linked to a promoter sequence; andregenerating a transgenic plant from the transformed explant, thetransgenic plant having the at least 10 transgenes arranged in tandem inthe single locus, wherein each of the at least 10 transgenes areexpressed at levels sufficient to confer a selectable or screenablemarker phenotype or a trait of agronomic interest to the transgenicplant.
 16. The method of claim 15, wherein expression of at least two ofthe transgenes of the plant is enhanced relative to expression of thesame transgenes in an otherwise isogenic plant having fewer than 10transgenes.
 17. The method of claim 15, wherein at least one of thetransgenes transformed into the single locus confers a trait ofagronomic interest to the transgenic plant.
 18. The method of claim 17,wherein the trait of agronomic interest conferred by the at least onetransgene is selected from the group consisting of herbicide tolerance,drought resistance, insect resistance, fungus resistance, virusresistance, bacteria resistance, male sterility, cold tolerance, salttolerance, increased yield, enhanced oil composition, increased oilcontent, enhanced nutrient use efficiency and altered amino acidcontent.
 19. The method of claim 15, wherein the single locus of thetransgenic plant comprises at least one transgene that confers herbicidetolerance and at least one transgene that confers insect resistance. 20.The method of claim 19, wherein the single locus of the transgenic plantcomprises at least two transgenes that confer herbicide tolerance and atleast two transgenes that confer insect resistance.
 21. The method ofclaim 19, wherein the single locus of the transgenic plant comprises atleast one transgene that confers above-ground insect resistance and atleast one transgene that confers below-ground insect resistance.
 22. Themethod of claim 15, wherein the transgenic plant is selected from thegroup consisting of wheat, maize, rye, rice, corn, oat, barley,turfgrass, sorghum, millet, sugarcane, tobacco, tomato, potato, soybean,cotton, canola, sunflower and alfalfa.
 23. The method of claim 19,wherein the single locus comprises at least one transgene that confersdrought tolerance.
 24. The method of claim 22, wherein the transgenicplant is a corn plant.